Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. In humans, the most common type of prion disease is Creutzfeldt-Jakob disease (CJD) which can occur in several forms. Sporadic CJD (sCJD) makes up the majority of the CJD cases and occurs randomly at an incidence of 1-2 per million people worldwide. Iatrogenic CJD (iCJD) is associated with exposure to prion contaminated medical instruments or products while familial CJD (fCJD) is associated with mutations in the prion protein gene. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. PrPSc can be deposited in the brain as either diffuse amyloid negative deposits or as dense amyloid positive deposits. For reasons that are not yet clear, amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unknown whether or not prion diseases where PrPSc is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrPSc is primarily deposited as non-amyloid. Multiple studies have shown that amyloid formed from amyloid beta (A) protein, alpha synuclein and tau can propagate via a prion-like mechanism and spread from cell-to-cell in transgenic mouse models e.g. Science 313: 1781-1784 (2006), Nat Cell Biol 11: 909-913 (2009), J Exp Med 209: 975-986 (2012). Based on these data, it has been suggested that amyloid formation in neurodegenerative proteinopathies such as Alzheimers Disease (AD) and Parkinsons disease (PD) occur via prion-like mechanisms and that proteins such as AD-associated A may also be transmissible, infectious prions. Co-deposition of misfolded proteins during neurodegeneration, such as the co-localization of PrPSc and A to plaques in some cases of sCJD ACTA Neuropathol 96:116-122 (1998), also suggest that interactions between these proteins could contribute to disease pathogenesis. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) Understanding the pathways of PrP amyloid formation and spread and, 2) Studying how mutations and amino acid polymorphisms in PrP influence PrPSc amyloid formation in familial forms of prion disease. Since PrPSc formation and spread appear to be mechanistically similar to the formation and spread of amyloid in other neurodegenerative diseases, the results of our prion studies will likely be broadly applicable to other diseases of protein misfolding and deposition. Different proteinase K (PK) cleavage sites in the N-terminus of PrPSc are indicative of differences in its structure. Based on the PK cleavage sites, two major structural forms of PrPSc have been identified in sCJD: Type 1 and Type 2. Recently, it has been found that many cases of sCJD are mixtures of Type 1 and Type 2 PrPSc suggesting that there may be a complex population of PrPSc molecules present with different secondary structures Brain 132: 2643 (2009). In 2018, we continued a project initiated in 2017 to use LC-MS/MS Nanospray Ion Trap Mass Spectrometry (MS) to precisely map the N-termini of PrPSc molecules associated with different neurological subtypes of CJD. We have currently completed about one-third of our experimental samples. The goal of this project is to determine whether certain structural populations of PrPSc correlate with specific CJD phenotypes. In 2018 we also continued a collaboration with Dr. Pedro Piccardo using MS to study BSE-infected non-human primates (NHP). These animals develop a neurodegenerative disease characterized by accumulation of PrPSc, hyper-phosphorylated tau, and alpha synuclein J Gen Virol 95:1612-16-18 (2014). In 2018, we used an MS protocol developed during the 2017 Annual Report period to obtain high-resolution protein identifications from our samples. The proteomics data we obtained will now be statistically analyzed and the results confirmed using non-MS based techniques. This experimental model will enable us to better understand the molecular mechanisms behind neurodegeneration in complex proteinopathies.